Anti-Ubiquitin (phospho S65) polyclonal antibody

The CRISPR/Cas9 system can be introduced into zebrafish as transgenes. Namely, expression of single-guide RNA (sgRNA) and controlled expression of Cas9 in transgenic zebrafish enables the study of gene functions in specific cell types. This transgenic CRISPR/Cas9 approach would be more useful if multiple sgRNAs could be expressed simultaneously since we could knock-out a gene more efficiently or disrupt multiple genes simultaneously. Here we describe a novel system to express multiple sgRNAs efficiently in zebrafish, that relies on the endogenous tRNA processing machinery. We cloned nine endogenous zebrafish tRNA genes, fused them to sgRNAs, and demonstrated that an active sgRNA can be produced from a precursor transcript containing either of these tRNAs. To show a proof of principle, we constructed transgenic fish expressing Cas9 under the control of the ubiquitin promoter and a single transcript containing three distinct sgRNAs, that targeted the slc45a2 (albino) gene, fused to tRNAs under the control of the U6 promoter. We found that the Tg(ubb:SpCas9, u6c:3xslc45a2-sgRNA) harbored mutations in all of the target sites in the albino gene and showed nearly complete albino phenotypes, which were amenable to imaging experiments. Thus, the tRNA-based multiplex sgRNA expression system should facilitate gene knock-out studies in transgenic zebrafish.

An "underbody blast" (UBB) is the detonation of a mine or improvised explosive device (IED) underneath a vehicle. In recent military conflicts, the incidence of UBBs has led to severe injuries, specifically in the lower extremities The foot and ankle complex, particularly the calcaneus bone, may sustain significant damage. Despite the prevalence of calcaneal injuries, this bone's unique properties and the progression of fracture and failure have not been adequately studied under high strain rate loading. This research discusses early efforts at creating a high-resolution computational model of the human calcaneus, with primary focus on modeling the fracture network through the complex microstructure of the bone and creating micromechanically-based constitutive models that can be used within full human body models. The ultimate goal of this ongoing research effort is to develop a micromechanics-based simulation of calcaneus fracture and fragmentation due to impact loading. With the goal of determining the basic mechanisms of stress propagation through the internal structure of the calcaneus, a two-dimensional model was employed for preliminary simulations with a plane-strain approximation. In this effort, a cadaveric calcaneus was scanned to a resolution of 55 mu m using an industrial micro-computed tomography (microCT) scanner. A mid-sagittal plane slice of the scan was selected and post-processed to generate a 2D finite element mesh of the calcaneus that included marrow, trabecular bone, and cortical bone elements. The calcaneus was modeled using two-dimensional quadratic plane strain elements. A fixed boundary condition was applied to the portion of the calcaneus that, in situ, would be restrained by the talus. A displacement of 1.25 mm was applied to the heel of the calcaneus over 5 ms. In a typical result, following impact, the strain and stress are propagated throughout the cortical shell and then began to radiate into the bone into the bone along the trabeculae. Local stress concentrations can be observed in the trabecular structure in the posterior region of the bone following impact. Upon impact, cortical and trabecular bone show different stresses of 13 MPa and 1 MPa, respectively, and exhibit complex high frequency responses. Observed results may offer insight into the wave interactions between the different materials comprising the calcaneus, such as impedance mismatch and refraction. Pore pressure in the marrow may be another important factor to consider in understanding stress propagation in the calcaneus.